Pharmaceutical composition

ABSTRACT

To provide a pharmaceutical composition which can enhance the storage stability of 1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole without impairing the effect of the compound. 
     The pharmaceutical composition includes 1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole, which is represented by formula (1): 
                         
and a compound having chelating ability.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.12/523,067, filed Jul. 14, 2009, which is a 371 of PCT/JP08/000040,filed on Jan. 17, 2008, and claims priority to Japanese PatentApplication No. 2007-016789, filed on Jan. 26, 2007.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition and, moreparticularly, to a pharmaceutical composition for use in cancerradiotherapy.

BACKGROUND ART

In cancer radiotherapy, 2-nitroimidazole derivatives are known to beuseful drugs which increase radiation sensitivity of cancer cells withradioresiatance in a hypoxic state, and enhance the effect ofradiotherapy. Among 2-nitroimidazole derivatives,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole, whichis represented by the following formula (1), has particularly highhydrophilicity and virtually no transferability to neurocytes, tothereby serve as a radiation sensitizer having no toxicity to thecentral nervous system (see, for example Patent Documents 1 to 3). Inaddition to exhibiting radiation sensitizing effect to hypoxic cells,the compound (1) has hydroxyl-group-removing action in nucleic acid(see, for example, Patent Document 4), apoptosis-signal maintainingaction (see, for example, Patent Document 5), etc. Thus, the compound(1) is considered to be a useful drug in cancer therapy.

In general, the action mechanism of a 2-nitroimidazole derivative isthought to include a re-oxidizing step attributed to very strongelectron affinity of the 2-position nitro group. However, such anelectron affinity of the 2-nitro group also means the instability of thenitro group. Actually, the nitro group is readily decomposed to formnitrogen monoxide or other species. It has also been known that sidechain cleavage occurs under acidic conditions, leading to release of2-nitroimidazole. In other words, stability of 2-nitroimidazolederivatives is thought to be impaired by the 2-position nitro group,which is a target-action-providing group. Therefore, there has beendemand for the development of means for enhancing storage stability of2-nitroimidazole derivatives without impairing the effect thereof.

Meanwhile, several means for stabilizing compounds having a nitro groupare known. For example, the means include stabilization by phospholipid(see, for example, Patent Document 6) and stabilization by a buffer suchas citric acid-citrate salt or phosphoric acid-phosphate salt (see, forexample, Patent Document 7). It has also been known that a chelatingagent exhibits a positive effect on stabilization of nitro compounds(see, for example, Patent Document 6). However, there has not been knownmeans for stabilizing the aforementioned 2-nitroimidazole derivatives.Particularly, it has never been known how a chelating agent stabilizes2-nitroimidazole derivatives.

-   Patent Document 1: JP-A-1991-223258-   Patent Document 2: WO 1994/014778-   Patent Document 3: JP-A-2003-321459-   Patent Document 4: JP-A-2005-27515-   Patent Document 5: JP-A-1997-77667-   Patent Document 6: Japanese kohyo Patent Publication No. 2003-512430-   Patent Document 7: JP-A-1995-126017

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention has been accomplished under such circumstances.Thus, an object of the present invention is to provide a pharmaceuticalcomposition which can enhance the storage stability of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazolewithout impairing the effect of the compound.

Means for Solving the Problems

In view of the foregoing, the present inventors have carried outextensive studies, and have found that, through adding a compound havingchelating ability to1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole,decomposition of the 2-nitro group and release of 2-nitroimidazole canbe prevented, while the target effect of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole ismaintained. The present invention has been accomplished on the basis ofthis finding.

Accordingly, the present invention provides a pharmaceutical compositioncomprising1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole, whichis represented by formula (1):

and a compound having chelating ability.

The present invention also provides use of the composition for producinga radiation sensitizer.

The present invention also provides a cancer radiotherapy, characterizedby administering the composition in an effective amount to a subject inneed thereof and irradiating the subject.

EFFECTS OF THE INVENTION

According to the present invention, the storage stability of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole can beenhanced without impairing the effect thereof, whereby highly effectivecancer radiotherapy can be realized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A graph showing the results of the severe test carried out inExample 1.

FIG. 2 A graph showing the results of the severe test carried out inExample 2.

FIG. 3 A graph showing the results of the severe test carried out inExample 3.

FIG. 4 A graph showing the results of the severe test carried out inExample 4.

FIG. 5 A graph showing the results of the severe test carried out inExample 5.

FIG. 6 A graph showing the results of the severe test carried out inExample 6.

FIG. 7 A graph showing the results of the severe test carried out inExample 7.

FIG. 8 A graph showing the results of the severe test carried out inExample 8.

FIG. 9 A graph showing the results of the severe test carried out inExample 9.

FIG. 10 A graph showing the results of the severe test carried out inComparative Example 1.

FIG. 11 A graph showing the radiation sensitization effects of thetested pharmaceutical compositions.

BEST MODES FOR CARRYING OUT THE INVENTION

1-(1-Hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole(compound (1)) employed in the present invention includes fourstereoisomers: RS form, SR form, RR form, and SS form. In the presentinvention, each of the optically active substances or a mixture thereofsuch as a racemic mixture of species of the optically active substancesmay be used. From the viewpoint of efficacy, particularly preferred is aracemic mixture of an isomer having a stereo-structure represented byformula (2) (SR form) and an isomer having a stereo-structurerepresented by formula (3) (RS form).

In clinical tests, compound (1) is required to be stored at 5° C. beforetests in order to ensure stability of the compound. Thus, considerabledifficulty is encountered in handling compound (1), which must be storedunder the above conditions. Compound (1) may be produced through amethod disclosed in Patent Document 1 or 2. In one specific productionprocedure, 2-nitro-1-trimethylsilylimidazole is condensed with2-acetoxymethoxy-1,3,4-triacetoxybutane in the presence of a Lewis acid,followed by deacetylation through reaction with sodium methoxide or asimilar substance. In this case, stereo-characteristics of2-acetoxymethoxy-1,3,4-triacetoxybutane are maintained in the finalproduct,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole.

The content of compound (1) in the pharmaceutical composition of thepresent invention is preferably 1 to 10 mass %, more preferably 2 to 8mass %. When the compound (1) content is excessively small, the amountof 1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole inthe produced pharmaceutical formulation is insufficient, impeding theeffect of compound (1), whereas when the amount is excessively large, insome cases a portion of the compound cannot be dissolved in a vehicle,impairing the stability of the formulation.

No particular limitation is imposed on the compound having chelatingability employed in the present invention, so long as the compound canbe employed as an additive for pharmaceutical products. Examples of suchcompounds include aldonic acids such as gluconic acid and glucepticacid; aldaric acids such as tartaric acid and glucaric acid; uronicacids such as glucuronic acid; saccharic acids or a salt thereof such asascorbic acid; aminopolycarboxylic acids or a salt thereof such asedetic acid and pentetic acid; oxycarboxylic acids or a salt thereofsuch as citric acid; amine compounds such as diethylamine; andpolyoxyethylene castor oils such as polyoxyl 35 castor oil. Of these,saccharic acids or a salt thereof, aminopolycarboxylic acids or a saltthereof, and amine compounds are preferred, with tartaric acid or a saltthereof, ascorbic acid or a salt thereof, pentetic acid or a saltthereof, and diethylamine being particularly preferred.

Examples of preferred salts include alkali metal salts such as sodiumsalts and potassium salts; alkaline earth metal salts such as calciumsalts and magnesium salts; organic amine salts such as ammonium salts,triethylamine salts, triethanolamine salts, and monoethanolamine salts;and basic amino acid salts such as lysine salts and alginic acid salts.These compounds having chelating ability may be employed in thecomposition singly or in combination of two or more species.

The amount of compound having chelating ability contained in thepharmaceutical composition of the invention is preferably 0.005 to 5mass %, more preferably 0.01 to 2 mass %. When the amount of compoundhaving chelating ability is excessively small, stability-enhancingeffect may fail to be attained, whereas when the amount is excessivelylarge, the stabilization effect attributed to addition reach a plateau,and formulation of interest may fail to be prepared.

Specifically, when a saccharic acid or a salt thereof is employed, theamount is preferably 0.01 to 2 mass %, more preferably 0.025 to 1.1 mass%. When an aminopolycarboxylic acid or a salt thereof such as edeticacid or a salt thereof, pentetic acid, or calcium trisodium pentetate isemployed, the amount is preferably 0.01 to 2 mass %, more preferably0.01 to 1.0 mass %. Particularly when pentasodium pentetate is employed,the amount is preferably 0.01 to 0.8 mass %, more preferably 0.01 to 0.5mass %. When an oxycarboxylic acid or a salt thereof is employed, theamount is preferably 0.01 to 2 mass %, more preferably 0.02 to 1 mass %.When an amine compound is employed, the amount is preferably 0.01 to 0.1mass %, more preferably 0.01 to 0.08 mass %. When a polyoxyethylenecastor oil is employed, the amount is preferably 0.01 to 0.8 mass %,more preferably 0.01 to 0.5 mass %.

The pharmaceutical composition of the present invention is preferablyemployed as a drug, particularly for the purpose of cancer therapy,inter alia, enhancing radiation sensitivity of hypoxic cancer cells incancer radiotherapy. Examples of the cancer to be preferably treated bythe pharmaceutical composition include lung cancer and pancreaticcancer. In a specific procedure of cancer radiotherapy, the compositionof the present invention is administered to a patient, and the patientis irradiated.

No particular limitation is imposed on the dosage form of thepharmaceutical composition, and the form may be appropriately selectedin accordance with the therapeutic purpose. Examples of the dosage forminclude oral forms such as tablets, capsules, granules, film-coateddrugs, powder, and syrup; and parenteral forms such as injectionformulation, suppositories, inhalation, percutaneously absorbableagents, eye drops, and nasal drops. Among them, injection formulation ispreferred from the viewpoint of rapid metabolism. The type of injectionformulation includes formulations for dripping. A dripping formulationis preferred for the following reason. The dose of a formulationcontaining1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole, aneffective ingredient, is increased, since a large amount of theeffective ingredient must be administered. In this case, a single doseadministration may cause a risky state of the relevant patient. Noparticular limitation is imposed on the form of such an injectionsolution, and it may be a solution or a freeze-dry formulation. However,a solution is preferred, since a satisfactory solubility can beattained. Example of preferred vehicles for the solution include purewater, physiological saline, and glucose solution which may beisotonified.

The pharmaceutical composition of the present invention may furthercontain any of the formulation ingredients generally employed inpreparation of drugs, so long as the effect of the present invention isnot impaired. Examples of such optional ingredients include polyhydricalcohols such as Macrogol; tonicity agents such as sodium chloride;buffer salts such as phosphate salt; excipients such as crystallinecellulose and starch; nonionic surfactants such aspolyoxyethylene-hardened castor oil; anionic surfactants such as sodiumlauryl sulfate; viscosity-increasing polysaccharides such as gum arabic;lubricants such as magnesium stearate; colorants; flavoringagents/deodorants; binders such as hydroxypropyl cellulose; and coatingagents such as “Eudragit” (registered trademark). In the case of aninjection liquid, a particularly preferred formulation contains onlycompound (1), a compound having chelating ability, and a vehicle, anddoes not contain other ingredients.

The pharmaceutical composition of the present invention may be producedthrough processing the aforementioned essential ingredients and optionalingredients in a routine manner.

The dose of the pharmaceutical composition of the present invention maybe appropriately selected in consideration of the body weight, age, sex,symptom, etc. of a patient. Generally, the daily dose for an adult ispreferably 1 to 10 g as1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1

Solutions for drip infusion (i.e., pharmaceutical compositions 1 to 6),which are pharmaceutical compositions of the present invention, wereprepared according to the below-described formulations. Specifically,the formulated ingredients were weighed and placed in a container, andthe mixture was stirred for dissolution. Each of the formulated productswas stored for 9 or 10 days under severe conditions (55° C.), and thecontent of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 1 shows the results.

As is clear from FIG. 1,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 1 Ingredients (mass %) Compound (1) (racemic)  5 Chelatingcompound amount in Table 2 Water balance Total 100

TABLE 2 (mass Samples Chelating compound %) Pharmaceutical compn. 1Calcium saccharate*¹ 0.3 Pharmaceutical compn. 2 Calcium disodiumedetate 0.04 Pharmaceutical compn. 3 Calcium gluconate 0.025Pharmaceutical compn. 4 Magnesium gluconate 1.1 Pharmaceutical compn. 5Trisodium citrate 0.9 Pharmaceutical compn. 6 Disodium hydrogencitrate0.02 Control 0.0 *¹Calcium saccharate: defined in JapanesePharmaceutical Excipients

Example 2

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 7 to 10), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 2 shows the results.

As is clear from FIG. 2,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 3 Ingredients (mass %) Compound (1) (racemic)  5 Calcium trisodiumpentetate amount in Table 4 Water balance Total 100

TABLE 4 Calcium Samples trisodium pentetate (mass %) Pharmaceuticalcompn. 7 0.01 Pharmaceutical compn. 8 0.05 Pharmaceutical compn. 9 0.1Pharmaceutical compn. 10 1.0 Control 0.0

Example 3

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 11 to 14), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 3 shows the results.

As is clear from FIG. 3,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 5 Ingredients (mass %) Compound (1) (racemic)  5 Pentasodiumpentetate amount in Table 6 Water balance Total 100

TABLE 6 Samples Pentasodium pentetate (mass %) Pharmaceutical compn. 110.01 Pharmaceutical compn. 12 0.05 Pharmaceutical compn. 13 0.1Pharmaceutical compn. 14 1.0 Control 0.0

Example 4

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 15 to 17), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 4 shows the results.

As is clear from FIG. 4,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 7 Ingredients (mass %) Compound (1) (racemic)  5 Diethylamineamount in Table 8 Water balance Total 100

TABLE 8 Samples Diethylamine (mass %) Pharmaceutical compn. 15 0.01Pharmaceutical compn. 16 0.05 Pharmaceutical compn. 17 0.1 Control 0.0

Example 5

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 18 to 21), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 5 shows the results.

As is clear from FIG. 5,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 9 Ingredients (mass %) Compound (1) (racemic)  5 Polyoxyl 35castor oil amount in Table 10 Water balance Total 100

TABLE 10 Samples Polyoxyl 35 castor oil (mass %) Pharmaceutical compn.18 0.01 Pharmaceutical compn. 19 0.05 Pharmaceutical compn. 20 0.1Pharmaceutical compn. 21 1.0 Control 0.0

Example 6

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 22 to 24), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 6 shows the results.

As is clear from FIG. 6,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 11 Ingredients (mass %) Compound (1) (racemic)  5 Sodium ascorbateamount in Table 12 Water balance Total 100

TABLE 12 Samples Sodium ascorbate (mass %) Pharmaceutical compn. 22 0.01Pharmaceutical compn. 23 0.05 Pharmaceutical compn. 24 0.1 Control 0.0

Example 7

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 25 to 28), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazolecontent of the product was periodically quantitated. As a control, a 5mass % aqueous solution of compound (1) was employed. FIG. 7 shows theresults.

As is clear from FIG. 7,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 13 Ingredients (mass %) Compound (1) (racemic)  5 Sodium tartratedihydrate amount in Table 14 Water balance Total 100

TABLE 14 Samples Sodium tartrate dihydrate (mass %) Pharmaceuticalcompn. 25 0.01 Pharmaceutical compn. 26 0.05 Pharmaceutical compn. 270.1 Pharmaceutical compn. 28 1.0 Control 0.0

Example 8

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 29 to 32), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 8 shows the results.

As is clear from FIG. 8,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 15 Ingredients (mass %) Compound (1) (racemic)  5 Calciumgluceptate amount in Table 16 Water balance Total 100

TABLE 16 Samples Calcium gluceptate (mass %) Pharmaceutical compn. 290.01 Pharmaceutical compn. 30 0.05 Pharmaceutical compn. 31 0.1Pharmaceutical compn. 32 1.0 Control 0.0

Example 9

In a manner similar to that of Example 1, solutions for drip infusion(i.e., pharmaceutical compositions 33 to 36), which are pharmaceuticalcompositions of the present invention, were prepared according to thebelow-described formulations. Each of the formulated products was storedfor 8 days under severe conditions (55° C.), and the content of1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole in theproduct was periodically quantitated. As a control, a 5 mass % aqueoussolution of compound (1) was employed. FIG. 9 shows the results.

As is clear from FIG. 9,1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazole hasbeen found to be stable in the pharmaceutical compositions of thepresent invention, even under severe conditions.

TABLE 17 Ingredients (mass %) Compound (1) (racemic)  5 Sodiumgluceptate amount in Table 18 Water balance Total 100

TABLE 18 Samples Sodium gluceptate (mass %) Pharmaceutical compn. 330.01 Pharmaceutical compn. 34 0.05 Pharmaceutical compn. 35 0.1Pharmaceutical compn. 36 1.0 Control 0.0

Comparative Example 1

The procedure of Example 1 was repeated, except that a phosphate salthaving only a buffer action was used instead of a compound havingchelating ability, to thereby prepare solutions for drip infusionaccording to the below-described formulations. As a control, a 5 mass %aqueous solution of compound (1) was employed. The pharmaceuticalformulations were subjected to a storage test under sever conditions(55° C.) for 10 days. The results are shown in FIG. 10.

As is clear from FIG. 10, the composition containing a phosphate saltexhibited a compound (1) content of less than 95 mass % on day 9,indicating the effect of a compound having chelating ability. Therefore,the effect of the pharmaceutical composition of the present inventionwas found to be attained not by a buffer action of the salt but by achelating action.

TABLE 19 Ingredients (mass %) Compound (1) (racemic) 5 Sodiumdihydrogenphosphate*¹ 0.25 Water balance Total 100 *¹dihydrate

Test Example 1

The radiation sensitization effect of the present invention wasinvestigated through the micronucleus method by use of mouse squamouscell carcinoma cells (SCCVII).

Specifically, a gas (95% N₂+5% CO₂) was caused to pass through mousesquamous cell carcinoma cells (SCCVII) for 20 minutes, to thereby renderthe cells in a hypoxic state. Subsequently, in the presence of PBS and atest pharmaceutical formulation, the cells were irradiated with X-rays(0, 1, 2, or 3 Gy). The following tested pharmaceutical formulationswere employed: pharmaceutical composition 10 (Example 2), pharmaceuticalcomposition 12 (Example 3), pharmaceutical composition 15 (Example 4),pharmaceutical composition 21 (Example 5), pharmaceutical composition 23(Example 6), pharmaceutical composition 28 (Example 7), pharmaceuticalcomposition 32 (Example 8), and pharmaceutical composition 36 (Example9). A 5 mass % aqueous solution of compound (1) (racemic) was employedas a control. After irradiation, the cells were washed and cultured forabout 24 hours in the presence of cytochalasin B, to thereby generatebinucleate cells. The thus-treated cells were fixed and fluoro-stained,and the number of binucleate cells and that of micronuclei were counted,whereby occurrence of micronucleus generation was determined. FIG. 11shows the results.

In FIG. 11, “A” denotes control, “D” denotes pharmaceutical composition15, “I” denotes pharmaceutical composition 23, “M” denotespharmaceutical composition 28, “N” denotes pharmaceutical composition12, “O” denotes pharmaceutical composition 10, “T” denotespharmaceutical composition 21, “U” denotes pharmaceutical composition32, and “V” denotes pharmaceutical composition 36. In non-irradiatedgroups, occurrence of micronucleus generation was not changed by PBS orany of the tested formulations. No direct effect of the formulations onthe cells was observed. In other words, direct toxicity to the cells bythe additives was not observed. In contrast, in irradiated groups, alltested formulations exhibited radiation sensitization effect in ahypoxic state. In particular, pharmaceutical compositions 10, 12, 15,23, and 28 did not vary the sensitization effect, as compared with acontrol containing no compound having chelating ability.

The invention claimed is:
 1. A cancer radiotherapeutic method, whichcomprises: administering to a subject in need thereof, an effectiveamount of a pharmaceutical composition consisting essentially of a1-(1-hydroxymethyl-2,3-dihydroxypropyloxymethyl)-2-nitroimidazolecompound represented by formula (1):

and a compound having chelating ability, and subsequently irradiatingthe subject; wherein the compound having chelating ability is at leastone species selected from the group consisting of a saccharic acid, asalt of a saccharic acid, an aminopolycarboxylic acid, a salt of anaminopolycarboxylic acid, an oxycarboxylic acid, a salt of anoxycarboxylic acid, an amine compound, and a polyoxyethylene castor oil.2. The method of claim 1, wherein the compound having chelating abilityis at least one species selected from the group consisting of, a salt ofa saccharic acid, a salt of an aminopolycarboxylic acid, a salt of anoxycarboxylic acid, diethylamine, and a polyoxyethylene castor oil. 3.The method of claim 2, wherein the compound having chelating ability isan alkaline earth metal salt of gluconic acid, gluceptic acid, tartaricacid, glucuronic acid, glucaric acid, or ascorbic acid.
 4. The method ofclaim 1, wherein the compound represented by formula (1) is an RS.SRracemic mixture of an isomer having a stereo-structure represented byformula (2):

and an isomer having a stereo-structure represented by formula (3):


5. The method of claim 1, wherein the administering is performed throughinjection.
 6. The method of claim 1, wherein the pharmaceuticalcomposition contains the compound represented by formula (1) in anamount of 1 to 10 mass %.
 7. The method of claim 1, wherein thepharmaceutical compositions contains the compound represented by formula(1) in an amount of 2 to 8 mass %.
 8. The method of claim 1, wherein thecompound having chelating ability comprises diethylamine.
 9. The methodof claim 1, wherein the compound having chelating ability is a saccharicacid or a salt of a saccharic acid present in the pharmaceuticalcomposition in an amount of 0.025 to 1.1 mass %; an aminopolycarboxylicacid or a salt of an aminopolycarboxylic acid present in thepharmaceutical composition in an amount of 0.01 to 1.0 mass %; anoxycarboxylic acid or a salt of an oxycarboxylic acid present in thepharmaceutical composition in an amount of 0.02 to 1 mass %; an aminecompound present in the pharmaceutical composition in an amount of 0.01to 0.1 mass %; or a polyoxyethylene caster oil present in thepharmaceutical composition in an amount of 0.01 to 0.8 mass %.
 10. Themethod of claim 1, wherein the compound having chelating ability ispresent in the pharmaceutical composition an amount of 0.01 to 2 mass %.11. The method of claim 1, wherein the compound having chelating abilityis a saccharic acid or a salt of a saccharic acid present in thepharmaceutical composition in an amount of 0.025 to 1.1 mass %.
 12. Themethod of claim 1, wherein the compound having chelating ability is anaminopolycarboxylic acid or a salt of an aminopolycarboxylic acidpresent in the pharmaceutical composition in an amount of 0.01 to 1.0mass %.
 13. The method of claim 1, wherein the compound having chelatingability is an oxycarboxylic acid or a salt of an oxycarboxylic acidpresent in the pharmaceutical composition in an amount of 0.02 to 1 mass%.
 14. The method of claim 1, wherein the compound having chelatingability is an amine compound present in the pharmaceutical compositionin an amount of 0.01 to 0.1 mass %.
 15. The method of claim 1, whereinthe compound having chelating ability is a polyoxyethylene caster oilpresent in the pharmaceutical composition in an amount of 0.01 to 0.8mass %.
 16. The method of claim 1, wherein the compound having chelatingability is edetic acid, a salt of edetic acid, pentetic acid, or a saltof pentetic acid.
 17. The method of claim 1, wherein the compound havingchelating ability is calcium trisodium pentetate or pentasodiumpentetate.
 18. The method of claim 1, wherein the compound representedby formula (1) is at least one member selected from the group consistingof the RS form, SR form, RR form, and SS form of the compoundrepresented by formula (1).
 19. The method of claim 18, wherein thecompound represented by formula (1) is an optically active stereoisomerselected from the group consisting of the RS form, SR form, RR form, andSS form of the compound represented by formula (1).